Many diseases are caused by known genetic alterations or mutations that occur in specific, identified regions in the DNA of a patient. Detection of a disease by direct analysis of DNA offers significant advantages in certainty and accuracy over other diagnostic techniques but requires an assay device and methodology that accurately detects and identifies the genetic alterations in a patient sample. When specific disease-related alterations or mutations can be detected and identified as markers of a disease, the DNA-based assays can be used both to diagnose an individual patient for disease and to screen patient groups for the presence of known genetic markers that may be correlated with disease. In some cases, populations of patients are uniquely susceptible to certain diseases or groups of diseases and DNA-based assays can be used to screen patients for any number of a group of diseases based on the detection of mutations in a patient's DNA.
In recent years, scientists have developed numerous techniques to analyze genetic material. Together with research that uncovered the specific genetic markers underlying certain diseases, researchers have developed techniques to detect the presence of specific alterations occurring in identified regions in a patient's DNA. Several analytical techniques are available to detect genetic alterations when present, however, the detection of small differences within the entirety of a patient's DNA requires a sophisticated test regimen and requires highly specialized biochemical reagents. For this and other reasons, much of the instrumentation and testing methods can only be performed in research environments and requires highly skilled technicians to conduct the analyses and interpret the results. Also, the testing of subtle genetic alterations is often time consuming and expensive to perform. The situation is complicated by the fact that many diseases have dozens of potential underlying genetic factors that play a role in the onset or progression of the disease, and as the number increases, the cost and complexity of an assay to test a patient's genetic material substantially increases.
In a clinical environment, a single patient can be tested for the presence of a large number of mutations or polymorphisms potentially underlying a suspected disease. When a screening approach is desired, the design of the assay technique becomes even more critical. If available, a screening assay would be highly desirable in several circumstances, including, to analyze the genetic basis for disease by detecting polymorphisms in patients and correlating the results to the presence, absence, or onset of one or more diseases, to screen susceptible groups of patients for genetic markers that exist for any of a group of diseases that are known to be passed from parent to child within ethnic groups, and to locate asymptomatic carriers of diseases who can pass the underlying mutation to offspring. An assay that is useful for a screening must be sufficiently reliable and cost-effective so that multiple tests can be efficiently performed on a large patient group.
For screening, an ideal testing system would be automated and capable of screening a large number of patient samples simultaneously and determining whether any one of a large number of mutations is present. Such a “high throughput” system would also require specially designed data processing so that assay results could be efficiently processed, correlated to patient data, and presented in a useful format for interpretation by clinical laboratory personnel. For analyzing patient samples, it is often desirable to test a large number of samples to first determine whether any one of a set of genetic markers is present, followed by analyzing individual samples to determine which member of the set of markers is present. In this fashion, multiple samples are screened to identify patients who are “positive” for a member of a set of markers, followed by identifying the specific mutation or polymorphism in the patient sample that yielded the positive signal. Furthermore, many diseases feature one or more of a small number of predominant mutations that occur with very high frequency. The existence of one or more predominant mutations may dictate that a testing assay should separately analyze selected mutations individually in a patient sample. Thus, the ideal screening and assay system would rapidly indicate, for an individual patient, whether or not any one or more of a set of known markers are present and would then offer the capability to identify, when a positive signal was generated in this screening process, the specific mutation or polymorphism that yielded the positive signal from among the larger set tested in the screening process.
Currently, a number of different techniques exist for direct analysis of a patient DNA sample. In one technique, synthetic strands of DNA are produced that have sequences that may or may not contain a mutation that are complementary to a select group of mutations and that can be used as probes to detect the mutation in a patient sample. These synthetic sequences are exposed to a patient sample, and when the mutation is present, the synthetic DNA becomes attached to the patient's DNA by hybridization. Once hybridized, the probe can be detected by several known techniques. Also, specific segments of patient DNA that may or may not contain a mutation can be amplified and the amplified DNA can be localized on an electronic microchip for further testing.
Cystic fibrosis (CF) is an example of a genetic disease that is caused, individually or collectively, by any of a number of different mutations. Cystic fibrosis afflicts approximately 30,000 children and adults in the United States; afflicted patients typically die in their thirties. One in 31 Americans (one in 28 Caucasians)—more than 10 million people—is an unknowing, symptom-free carrier of a mutation that leads to the disease. An afflicted patient must have inherited two defective copies of a specific gene—one from each parent—to have CF. Each time two CF carriers conceive a child, there is a 25 percent chance that the child will have CF, a 50 percent chance that the child will be an asymptomatic carrier; and a 25 percent chance that the child will be a non-carrier.
CF has a variety of symptoms that are manifested clinically. CF causes the body to produce an abnormally thick sticky mucus, due to the faulty transport of sodium chloride (salt) within cells lining organs such as the lungs and pancreas, to their outer surfaces. The thick CF mucus also obstructs the pancreas, preventing enzymes from reaching the intestines to help break down and digest food. CF patients also suffer from persistent coughing, wheezing or pneumonia; excessive appetite but poor weight gain and bulky stools. The sweat test is a common diagnostic test for CF. This test measures the amount of salt in the skin and a high salt level indicates that a person has CF.
The treatment of CF depends upon the stage of the disease and which organs are involved. One measure of treatment, chest physical therapy, requires vigor percussion (by using cupped hands) on the back chest to dislodge the thick mucus from the lungs. Antibiotics are also used to treat lung infections administered intravenously, via pills, and/or medical vapors that are inhaled to open up clogged airways. When CF affects the digestive system, the body does not absorb enough nutrients. Therefore, people with CF may need to eat an enriched diet and take both replacement vitamins and enzymes.
CF is known to be caused by a large number of mutations, at least 25 have been identified as major contributors to the disease. In August 2001, the American College of Gynecologists (ACOG) recommended testing the general group of potential parents for the 25 separate genetic markers to identify asymptomatic carriers who risk passing the disease to children. Because of the need to screen a large group of patients, a test for CF should rapidly and accurately screen multiple patient samples for the presence of any one or a set of known markers followed by the identification of one or more specific markers in those patients who test positive for at least one member of the set. Detection of whether or not any single one or more of the mutations exists provides a rapid screening method, and the detection and identification of the single mutation or number of mutations in a patient allows diagnosis of the disease or identification of a patient as a potential carrier.
In such situations, the design of the assay and methodology that efficiently achieves the goals described above is critical. Specifically, the assay must be rapid, accurate, and cost effective such that the assay can be performed as a routine part of patient care thereby expanding the utility of the assay from diagnosing individual patients to screening entire groups. The assay should be able to rapidly test multiple patient samples and be flexible enough to selectively recognize predominant mutations or markers for a disease. Through the ability to screen and identify a large number of genetic polymorphisms, the assay could both diagnose disease as well as yield epidemiological data about the prevalence of specific polymorphisms and the relation to the existence or severity of a condition that may be correlated to a specific disease or that exists in a number of pathologies. Because many diseases have underlying genetic markers that have been identified and localized to identified regions of a patient's DNA that can be analyzed, once the specific genetic markers are identified, any number of diseases can be analyzed using the same assay format by simply altering the gene specific reagents in the assay that hybridize with a patient's DNA to detect the known marker and correlating the presence of the marker with one or more diseases. Accordingly, once the assay design and methodology are realized, one additional disease, a group of diseases, or a group of polymorphisms that are directly or indirectly correlated to several diseases, can be detected with the assay format. As the genetic bases of other diseases are discovered, the gene specific assay reagents are readily modified to take advantage of the existing format to detect and analyze new diseases. For example, while cystic fibrosis is susceptible of detection by screening an identification of a discrete set of markers or mutations that are known to contribute to the disease, in other circumstances, the screening process may identify other polymorphisms that are not directly related to a single disease, but that are related to multiple diseases or that accompany different conditions such as a panel of diseases that may affect a certain population group.